The ubiquitin-proteasome system is believed to play a vital role in regulating proteins involved in maintaining genomic integrity. Genetic and biochemical studies in yeast and mammals have demonstrated that several ubiquitin conjugating enzymes and ubiquitin protein ligases including Ube2A, Ube2B, Ube2rE, Ube2N, BRCA1/BARD1, CDH1/APC are essential for the cellular response to DNA damage. However, the physiological substrates for these enzymes remain unknown. We have recently developed a unique signaling-based functional proteomic system that can specifically identify genome-wide ubiquitin-degraded proteins in response to DNA damage. This novel strategy will lead to the identification of substrates for these critical enzymes involved in the surveillance of genomic integrity. In addition, this combinatorial approach will result in the discovery of new regulatory ubiquitin components pivotal to the control and progression of cell cycle, initiation of transcription, DNA repair and apoptosis in response to genotoxic stress. The goal of this proposal is (1) to develop a large-scale in vitro protein expression technology that allows the synthesis of approximately 30,000 full-length proteins and (2) to apply this newly developed technology to identify genome-wide ubiquitin-degraded proteins in response to DNA damage signaling and (3) to characterize the role of identified proteins in maintaining genomic integrity. Taken together, the proposed work will test the hypothesis that ubiquitin-mediated circuitry is essential to ensure genomic stability. This investigation should fill significant gaps in the DNA damage checkpoint signaling pathway, and provide important insights into genomic stability regulated by ubiquitin. Given the importance of proteolytic regulation in human diseases, information gained from the proposed studies will be of direct relevance to biomedicine, including the identification of novel targets for biochemical agents that could interfere with the regulation.